Quicklinks

Related Product Information

Introduction

DETACHaBEAD® Mouse CD4 is intended for release of mouse CD4+ cells from Dynabeads after positive isolation with Dynabeads® Mouse CD4 (L3T4), (available separately, Cat. no. 114.45D). The released mouse CD4+ cells are pure, viable, not activated by the release-step and have no Dynabeads® or primary antibody bound to the surface.

Principle of Release

Release of Dynabeads® from the cells using DETACHaBEAD® provides a fast and reliable method of positive isolation. The cells may be isolated from mouse spleen, thymus or lymph nodes using Dynabeads® Mouse CD4 (L3T4) (not included). DETACHaBEAD® Mouse CD4 is then added to the bead-bound cells and will release the Dynabeads® and primary antibody from the mouse CD4+ T cells. Following a short incubation period, the released Dynabeads® are removed by magnetic separation and the positively selected cells collected. The isolated cells are pure (>99%), viable (>95%) and are not activated by the isolation or release procedure.
Downstream Application

The isolated cells are pure, viable and are free from antibody bound to the surface. The isolated cells can be use for any research applications, functional studies and flow cytometry. Cells positively isolated and released in this way have been shown to respond in a normal manner when stimulated with interleukin-2 or interferon-gamma.

Tubes: We recommend adapting the tube size to the working volume (e.g. using 1.5 ml Eppendorf tubes for small volumes).

Cell culture media: RPMI 1640/1% FCS (Foetal Calf Serum).

CRITICAL NOTES

Never resuspend the positively isolated cells in less than 100 μl Cell culture media, even if the starting sample is less than 1 x 107 MNC or 1 ml blood.

The volume of DETACHaBEAD® used relates to the volume of Dynabeads® used for cell isolation. Never use less than 10 μl DETACHaBEAD® per 25 μl Dynabeads® used for positive isolation. The amount of DETACHaBEAD® used can be directly scaled up.

Incubation at 2-8°C will significantly reduce the number of released cells. Incubating at 37°C will not increase release efficiency.

Protocol

The amount of DETACHaBEAD® needed to obtain optimal release varies with the number of Dynabeads® used. This protocol is based on an initial positive cell isolation step from a starting sample of 1 x 107 cells using 25 μl Dynabeads®. When working with less than 25 μl Dynabeads® for cell isolation, use the volumes indicated below for release. When working with > 25 μl Dynabeads®, scale up the release protocol accordingly.